Immunogenic bacterial vesicles with outer membrane proteins

ABSTRACT

Knockout of the meningococcal mltA homolog gives bacteria that spontaneously release vesicles that are rich in immunogenic outer membrane proteins and that can elicit cross-protective antibody responses with higher bactericidal titres than OMVs prepared by normal production processes. Thus the invention provides a bacterium having a knockout mutation of its mltA gene. The invention also provides a bacterium, wherein the bacterium: (i) has a cell wall that includes peptidoglycan; and (ii) does not express a protein having the lytic transglycosylase activity MltA protein. The invention also provides compositions comprising vesicles that, during culture of bacteria of the invention, are released into the culture medium.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the electronically submitted sequence listing (Name:VN51982_Seq_Lstg.txt; Size 39,612,036 bytes; and Dale of Creation: Aug.28, 2017) was originally submitted in U.S. application Ser. No.11/666,786 (now U.S. Pat. No. 9,206,399, filed Oct. 28, 2005) and isincorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention is in the field of vesicle preparation for immunisationpurposes.

BACKGROUND ART

One of the various approaches to immunising against N.meningitidisinfection is to use outer membrane vesicles (OMVs). An efficacious OMVvaccine against serogroup B has been produced by the Norwegian NationalInstitute of Public Health [e.g. ref. 1] but, although this vaccine issafe and prevents MenB disease, its efficacy is limited to thehomologous strain used to make the vaccine.

The ‘RIVM’ vaccine is based on OMVs containing six different PorAsubtypes. It has been shown to be immunogenic in children in phase IIclinical trials [2].

Reference 3 discloses a vaccine against different pathogenic serotypesof serogroup B meningococcus based on OMVs which retain a proteincomplex of 65-kDa. Reference 4 discloses a vaccine comprising OMVs fromgenetically-engineered meningococcal strains, with the OMVs comprisingat least one Class I outer-membrane protein (OMP) but not comprising aClass 2/3 OMP. Reference 5 discloses OMVs comprising OMPs which havemutations in their surface loops and OMVs comprising derivatives ofmeningococcal lipopolysaccharide (LPS).

As well as serogroup B N.meningitidis, vesicles have been prepared forother bacteria. Reference 6 discloses a process for preparing OMV-basedvaccines for serogroup A meningococcus. References 7 and 8 disclosevesicles from N.gonorrhoeae. Reference 9 discloses vesicle preparationsfrom N.lactamica. Vesicles have also been prepared from Moraxellacatarrhalis [10, 11], Shigella flexneri [12, 13], Pseudomonas aeruginosa[12, 13], Porphyromonas gingivals [14], Treponema pallidum [15],Haemophilus influenzae [16 & 21] and Helicobacter pylori [17].

The failure of OMVs to elicit cross-protection against non-homologousstrains is not well understood, particularly as most N.meningitidisisolates share a small number of conserved protective surface antigensthat, if present in OMVs, would be expected to provide broad protectivecoverage. One possible explanation for the failure is the existence ofvariable immune-dominant surface antigens that prevent the conservedantigens from exerting their protective action, and the presence ofimmune-dominant hyper-variable proteins such as PorA has beenextensively documented and demonstrated. Other possible explanations arethat the methods for OMV preparation result in contamination withcytoplasmic and/or inner membrane proteins that dilute the protectiveouter membrane proteins, or that antigens are lost by the detergentextraction.

There have been various proposals to improve OMV efficacy. Reference 18discloses compositions comprising OMVs supplemented with transferrinbinding proteins (e.g. TbpA and TbpB) and/or Cn,Zn-superoxide dismutase.Reference 19 discloses compositions comprising OMVs supplemented byvarious proteins. Reference 20 discloses preparations of membranevesicles obtained from N.meningitidis with a modified fur gene.Reference 21 teaches that nspA expression should be up-regulated withconcomitant porA and cps knockout. Further knockout mutants ofN.meningitidis for OMV production are disclosed in references 21 to 23.In contrast to these attempts to improve OMVs by changing expressionpatterns, reference 24 focuses on changing the methods for OMVpreparation, and teaches that antigens such as NspA can be retainedduring vesicle extraction by avoiding the use of detergents such asdeoxycholate.

It is an object of the invention to provide further and improved vesiclepreparations, together with processes for their manufacture. Inparticular, it is an object of the invention to provide vesicles whichretain important bacterial immunogenic components from N.meningitidis.

DISCLOSURE OF THE INVENTION

The invention is based on the surprising discovery that disruption ofthe pathways involved in degradation of peptidoglycan (the murein layer)gives bacteria that release vesicles into their culture medium, and thatthese vesicles are rich in immunogenic outer membrane proteins and canelicit broad-ranging bactericide immune responses. The vesicles aredifferent from the OMVs that can be prepared by disrupting wholebacteria (e.g. by sonication and sarkosyl extraction [25]), and can beprepared without even disrupting bacterial cells e.g. simply byseparating the vesicles from the bacteria by a process such ascentrifugation.

In particular, the inventors have found that knockout of themeningococcal mltA homolog (also referred to as ‘GNA33’ or ‘NMB0033’[26]) leads to the spontaneous release of vesicles that are rich inimmunogenic outer membrane proteins and that can elicit broadlycross-protective antibody responses with higher bactericidal titres thanOMVs prepared by normal production processes. This enhanced efficacy issurprising for two reasons: first, the NMB0033 protein has previouslybeen reported to be highly effective in raising bactericidal antibodies(e.g. see table 1 of ref. 196) and to be a strong vaccine candidate(e.g. see table 2 of ref. 27), with a recommendation in reference 28that it should be unregulated for vesicle production, so its loss woulda priori be expected to reduce bactericidal efficacy rather than toincrease it; second, the knockout strains do not have the correcttopological organisation of the cellular membrane, and the mainconstituent proteins of normal OMVs (e.g. the PorA, PIB, class 4 andclass 5 outer membrane proteins) had previously been reported to bereleased into culture medium [25]. The inventors have now found that thepreviously-reported release does not involve secretion of discreteproteins, but that instead the outer membrane proteins are released inthe form of vesicles. These vesicles are advantageous over OMVs preparedby prior art means because they are released spontaneously into theculture medium and can thus be prepared simply and efficiently withoutthe complicated and time-consuming disruption and purification methodsthat are normally used for preparing OMVs.

Thus the invention provides a bacterium having a knockout mutation ofits mltA gene. The bacterium preferably also has a knockout mutation ofat least one further gene e.g. the porA and/or porB and or IpxA genes.

The invention also provides a bacterium, wherein: (i) the bacterium hasa cell wall that includes peptidoglycan; and (ii) the bacterium does notexpress a protein having the lytic transglycosylase activity of MltAprotein. The bacterium is preferably a mutant bacterium i.e. thebacterium is a mutant strain of a wild-type species that expresses MltAprotein. The bacterium preferably also does not express at least onefurther protein e.g. the PorA and/or PorB and/or LpxA proteins.

Preferred bacteria of the invention are in the genus Neisseria, such asN.meningitidis, and so the invention provides a meningococcus bacteriumhaving a knockout mutation of its gna33 gene. A preferred meningococcusis gna33⁻ IpxA⁻ PorA⁻.

The invention also provides a composition comprising vesicles that,during culture of bacteria of the invention, are released into theculture medium. This composition preferably does not comprise any livingand/or whole bacteria. This composition can be used for vaccinepreparation.

The invention also provides a composition comprising vesicles, whereinthe vesicles are present in the filtrate obtainable after filtrationthrough a 0.22 μm filter of a culture medium in which a bacterium of theinvention has been grown. This composition can be used for vaccinepreparation.

The invention also provides a meningococcal vesicle, wherein the vesicledoes not include at least one of (i.e. does not include 1, 2 or 3 of)MinD, FtsA, and/or phosphoenolpyruvate synthase. The invention alsoprovides a meningococcal vesicle, wherein the vesicle does not includeat least one of NMB proteins 0126, 0154, 0157, 0171, 0219, 0359, 0387,0426, 0595, 0617, 0618, 0631, 0757, 0763, 0875, 0876, 0943, 0946, 0957,1131, 1252, 1323, 1341, 1445, 1497, 1574, 1576, 1869, 1934, 1936, 2096and/or 2101. The invention also provides a meningococcal vesicle,wherein the vesicle is substantially free from ribosomes. The inventionalso provides a meningococcal vesicle, wherein the vesicle issubstantially free from any aminoacid-tRNA-synthetases. The inventionalso provides a meningococcal vesicle, wherein the vesicle issubstantially free from any enzyme from the Krebs cycle. These vesicleswill also not include MltA (because of the knockout mutation), but willinclude outer membrane proteins. The vesicles may include trimeric outermembrane proteins (FIG. 13).

The invention also provides a meningococcal vesicle, which includes thefollowing 47 proteins: NMB0035, NMB0044, NMB0086, NMB0088, NMB0109,NMB0124, NMB0138, NMB0182, NMB0204, NMB0278, NMB0294, NMB0313, NMB0345,NMB0346, NMB0382, NMB0460, NMB0461, NMB0550, NMB0554, NMB0623, NMB0634,NMB0663, NMB0703, NMB0787, NMB0873, NMB0928, NMB1030, NMB1053, NMB1057,NMB1126, NMB1285, NMB1301, NMB1332, NMB1429, NMB1483, NMB1533, NMB1567,NMB1612, NMB1710, NMB1870, NMB1898, NMB1949, NMB1961, NMB1972, NMB1988,NMB2039 and NMB2091.

The invention also provides a meningococcal vesicle, which includes oneor more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, or 19) of the following 19 proteins: NMB0044, NMB0086, NMB0204,NMB0278, NMB0294, NMB0313, NMB0345, NMB0346, NMB0460, NMB0550, NMB0873,NMB0928, NMB1030, NMB1057, NMB1483, NMB1870, NMB1898, NMB1961, and/orNMB2091. See also Table 4 below.

The invention also provides a composition comprising a first set ofvesicles of the invention and a second set of vesicles of the invention,wherein said first and second sets are prepared from different strainsof meningococcus. The invention also provides a process for preparing amixture of vesicles, comprising: (a) preparing vesicles of the inventionfrom a first meningococcal strain; (b) preparing vesicles of theinvention from a second meningococcal strain; and (c) combining thevesicles from (a) and (b). Combining vesicles from different strains canimprove the coverage of clinical strains.

The invention also provides a process for preparing bacterial vesicles,comprising the steps of: (i) culturing a MltA⁻ bacterium in a culturemedium such that the bacterium releases vesicles into said medium; and(ii) collecting the vesicles from said medium. The MltA⁻ bacterium ispreferably a MltA knockout mutant. The vesicles can be collected by sizeseparation (e.g. filtration, using a filter which allows the vesicles topass through but which does not allow intact bacteria to pass through),which can conveniently be performed after centrifugation topreferentially pellet cells relative to the smaller vesicles (e.g. lowspeed centrifugation).

Peptidoglycan Metabolism

Peptidoglycan (also known as murein, mucopeptide or glycosaminopeptide)is a heteropolymer found in the cell wall of most bacteria.Peptidoglycan is the component that is primarily responsible for themechanical strength of the bacterial cell wall and for maintainingcellular shape. In Gram-positive bacteria it is the major component ofthe cell wall. In Gram-negative bacteria it occurs as a layer betweenthe cytoplasmic and outer membranes, and is covalently linked to theouter membrane via the Braun lipoprotein

Peptidoglycan consists mainly of linear heteropolysaccharide backbonechains that are cross-linked by ‘stem’ peptides to form a latticestructure. It is a polymer so large that it can be thought of as asingle immense covalently linked molecule. In E.coli the saccharidebackbone is formed from alternating N-acetylglucosamine (GlcNAc) andN-acetylmuramic acid (MurNAc) residues. A MurNAc residue may be linkedto a stem tetrapeptide. Cross-links between backbone chains are usuallyformed directly between D-alanine in one stem peptide and a meso-DAP ofanother. The E.coli structure is typical for Gram-negative bacteria, butthere is more variation within Gram-positive bacteria e.g. in S.aureus30-50% of the muramic acid residues are not acetylated, the stem peptideoften has L-lysine in place of meso-DAP and isoglutamine in place ofD-glutamate, and cross-links can occur between stem peptides.

The initial step in E.coli peptidoglycan biosynthesis is the formationof the UDP derivative of GlcNAc, which occurs in the cytoplasm. SomeUDP-GlcNAc is converted to UDP-MurNAc in a reaction of UDP-GlcNAc andphosphoenolpyruvate (PEP), catalysed by PEP:UDP-GlcNAc enolpyruvyltransferase. Still within the cytoplasm, amino acids are addedsequentially to UDP-MurNAc to form a UDP-MurNAc-pentapeptide known asthe ‘Park nucleotide’ that includes a terminal D-alanyl-D-alanine. ThePark nucleotide is then transferred to bactoprenol monophosphate in thecytoplasmic membrane, where UDP-GlcNAC is also added to make abactoprenol-disaccharide-pentapeptide subunit. Thedisaccharide-pentapeptide subunit is then transferred into theperiplasmic region, with bactoprenol-pyrophosphate remaining in themembrane. Within the periplasm the transferred subunit is inserted intoa growing peptidoglycan.

To allow cell division, changes in shape, and import/export of largecomplexes (e.g. during conjugation) then peptidoglycan degradation mustoccur. In E.coli this degradation is caused by enzymes referred to asmurein hydrolases [29], which as a family includes lytictransglycosylases (mltA, mltB, mltC, mltD, stl70, emtA), endopeptidases(pbp4, pbp7, mepA) and amidases (amiC). Muramidases such as lysozymecleave the same β-(1-4)-glycosidic linkages between MurNAc and GlcNAcresidues; unlike muramidases, however, the transglycosylases cleave theglycosidic bond with concomitant formation of 1,6-anhydromuramoylresidues (AnhMurNAc).

The standard peptidoglycan anabolic and catabolic pathways are thuswell-characterised, as are the minor variations and modifications thatoccur between bacteria. The enzymes are well-characterised, and proteinshave been readily annotated as being involved in the pathways when newbacterial genomic sequences have been published. The skilled person canthus easily determine the enzymes involved in the peptidoglycanmetabolic pathway for any given bacterium, can easily identify theenzymes involved, and can easily identify the genes encoding thoseenzymes.

The invention is based on the knockout of the mltA gene, which encodes amembrane-bound lytic transglycosylase. The MltA family is recognised inINTERPRO (entry ‘ipr005300’) and PFAM (entry ‘MltA’ or ‘PF03562’), andthe PFAM record lists MltA proteins in bacteria as diverse as Rhizobiumloti, Bradyrhizobium japonicum, Brucella melitensis, Brucella suis,Rhizobium meliloti, Agrobacterium tumefaciens, Zymomonas mobilis,Caulobacter crescentus, Yersinia pestis, Salmonella typhimurium,Buchnera aphidicola, Photorhabdus luminescens, Escherichia coli,Shigella flexneri, Salmonella typhi, Pseudomonas aeruginosa, Pseudomonasputida, Pseudomonas syringae, Coxiella burnetii, Vibrio cholerae, Vibriovulnificus, Vibrio parahaemolyticus, Haemophilus ducreyi, Pasteurellamultocida, Chromobacterium violaceum, Neisseria meningitidis, Neisseriagonorrhoeae, Bordetella parapertussis, Bordetella bronchiseptica,Bordetella pertussis, Nitrosomonas europaea, Ralstonia solanacearum,Synechococcus elongatus, Gloeobacter violaceus, and Leptospirainterrogans.

Preferred bacteria for MltA knockout are in the Neisseria genus, withN.meningitidis being the most preferred bacterium. The MltA gene inserogroup B N.meningitidis has been referred to in the literature as‘GNA33’ [25,26,196], and an example sequence has GenBank accessionnumber ‘AF226391.1’. The MltA gene in serogroup A (‘NMA0279’) hasGenBank accession number NP_283118.1. Aligned polymorphic forms ofmeningococcal MltA can be seen in FIGS. 7 and 18 of reference 30. Twofull genome sequences of N.meningitidis are available [31,32]. For anygiven strain of N.meningitidis, therefore, the skilled person will beable to identify the mltA gene. For meningococcus, the knocked-out mltAgene is preferably the gene which, in the wild-type strain, has thehighest sequence identity to SEQ ID NO: 1 herein. MltA is a lipoproteinin meningococcus [26].

Knockout of mltA can result in reduced virulence, abnormal cellseparation, abnormal cell morphology, undivided septa, double septa,cell clustering and sharing of outer membranes [25]. At the same time,however, the knockout mutation has surprisingly been found to givebacteria that can spontaneously produce vesicles that are immunogenicand enriched in outer membrane proteins.

Bacteria

The bacterium from which vesicles are prepared may be Gram-positive, butit is preferably Gram-negative. The bacterium may be from genusMoraxella, Shigella, Pseudomonas, Treponema, Porphyromonas orHelicobacter (see above for preferred species) but is preferably fromthe Neisseria genus. Preferred Neisseria species are N.meningitidis andN.gonorrhoeae.

Within N.meningitidis, any of serogroups A, C, W135 and Y may be used,but it is preferred to prepare vesicles from serogroup B. Whererelevant, the meningococcus can be of any serotype (e.g. 1, 2a, 2b, 4,14, 15, 16, etc.), of any serosubtype (P1.2; P1.4; P1.5; P1.5,2;P1.7,16; P1.7,16b; P1.9; P1.9,15; P1.12,13; P1.13; P1.14; P1.15;P1.21,16; P1.22,14; etc.) and of any immunotype (e.g. L1; L3,3,7; L10;etc.), and preferred bacteria include: B:4:P1.4; B:4:P1.15;B:15:P1.7,16. The meningococcus may be from any suitable lineage,including hyperinvasive and hypervirulent lineages e.g. any of thefollowing seven hypervirulent lineages: subgroup I; subgroup III;subgroup IV-1; ET-5 complex; ET-37 complex; A4 cluster; lineage 3. Theselineages have been defined by multilocus enzyme electrophoresis (MLEE),but multilocus sequence typing (MLST) has also been used to classifymeningococci [ref. 33] e.g. the ET-37 complex is the ST-11 complex byMLST, the ET-5 complex is ST-32 (ET-5), lineage 3 is ST-41/44, etc.

Preferred strains within serogroup B are MC58, 2996, H4476 and 394/98.In some embodiments of the invention, however, the meningococcus is notstrain MC58 and is not strain BZ232.

As well as having a knockout of mltA, the bacterium may have one or moreknockout mutations of other gene(s). To reduce pyrogenic activity, forinstance, the bacterium should have low endotoxin (LOS/LPS) levels, andthis can be achieved by knockout of enzymes involved in LPSbiosynthesis. Suitable mutant bacteria are already known e.g. mutantNeisseria [34,35] and mutant Helicobacter [36]. The IpxA mutant ofmeningococcus is preferred. Processes for preparing LPS-depleted outermembranes from Gram-negative bacteria are disclosed in reference 37.

In N.meningitidis, a preferred further knockout is the PorA class Iouter membrane protein. Advantageously, such knockouts will not displaythe immunodominant hypervariable stain-specific PorA protein, therebyfocusing a recipient's immune response on other antigens. In a specificaspect, the invention provides a N.meningitidis bacterium, comprisingboth a knockout mutation of MltA and a knockout mutation of PorA. Thebacterium can also carry further knockout mutations e.g. in LOS/LPSsynthetic pathways (e.g. IpxA), immunodominant variable proteins, PorB,OpA, OpC, etc.

As well as having knockouts of particular endogenous genes, thebacterium may express one or more genes that are not endogenous. Forexample, the invention may use a recombinant strain that expresses newgenes relative to the corresponding wild-type strain. Although it ispreferred to knockout PorA expression, in an alternative approach it ispossible to engineer a meningococcus to express multiple PorA subtypes(e.g. 2, 3, 4, 5 or 6 of PorA subtypes: P1.7,16; P1.5,2; P1.19,15;P1.5c,10; P1.12,13; and P1.7h,4 [e.g. refs. 38, 39]). Expression ofnon-endogenous genes in this way can be achieved by various techniquese.g. chromosomal insertion (as used for introducing multiple PorA genes[40]), knockin mutations, expression from extra-chromosomal vectors(e.g. from plasmids), etc.

As well as down-regulating expression of specific proteins, thebacterium may over-express (relative to the corresponding wild-typestrain) immunogens such as NspA, protein 287 [19], protein 741 [41],TbpA [18], TbpB [18], superoxide dismutase [18], etc.

The bacterium may also include one or more of the knockout and/orover-expression mutations disclosed in reference 16, 21-24 and/or 42-43.Preferred genes for down-regulation and/or knockout include: (a) Cps,CtrA, CtrB, CtrC, CtrD, FrpB, GalB, HtrB/MsbB, LbpA, LbpB, LpxK, Opa,Opc, PilC, PorA, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [16];(b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK,Opa, Opc, PhoP, PilC, PmrE, PmrF, PorA, SiaA, SiaB, SiaC, SiaD, TbpA,and/or TbpB [21]; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalB, LbpA,LpbB, Opa, Opc, PilC, PorA, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/orTbpB [42]; and (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorA, PorB,SiaD, SynA, SynB, and/or SynC [43].

For meningococcal compositions, the selection criteria of reference 44may be used.

Preferred vesicles are prepared from meningococci having one of thefollowing subtypes: P1.2; P1.2,5; P1.4; P1.5; P1.5,2; P1.5,c, P1.5c,10;P1.7,16; P17,16b; P1.7h,4; P1.9; P1.15; P1.9,15; P1.12,13; P1.13; P1.14;P1.21,16; P1.22,14. The meningococcus is preferably in serogroup B.

Vesicles may also be prepared from the Escherichia genus, such as fromthe E.coli species. E.coli strains have traditionally been classified aseither commensal or pathogenic, and pathogenic strains are thensub-classified as intestinal or extraintestinal strains. Classificationmay also be based on the ‘K’ antigens. The best-studied ‘K’ antigen is‘K1’, which is considered to be the major determinant of virulence amongthose strains of E.coli that cause neonatal meningitis. Vesicles of theinvention can be prepared from any of these E.coli strains, but arepreferably from a pathogenic strain, including an extraintestinalpathogenic (‘ExPBC’ [45]) strain, a uropathogenic (UPEC) strain or ameningitis/sepsis-associated (MNBC) strains. Genome sequences ofpathogenic strains are available in the databases under accessionnumbers AE005174, BA000007 and NC-004431. Rather than use a mltAknockout, it may be preferred to knockout one or more of the componentsof the E.coli Tol-Pal complex [46], such as tolA, tolQ, tolB, pal and/ortolR. Knockout of tolR is preferred. The meningococci do not have ahomolog of the Tol-Pal system.

Vesicle Compositions

The invention provides the vesicles that are spontaneously released intoculture medium by bacteria of the invention. These vesicles are distinctfrom the vesicles that can be prepared artificially from the samebacteria, such as the sarkosyl-extracted OMVs prepared in reference 25from ‘ΔGNA33’ meningococci. They are also distinct from microvesicles(MVs [47]) and ‘native OMVs’ (‘NOMVs’ [64]), although vesicles of theinvention seem to be more similar to MVs and NOMVs than tosarkosyl-extracted OMVs. The vesicles are also distinct from blebs,which are outer-membrane protrusions that remain attached to bacteriaprior to release as MVs [48,49].

The vesicles of the invention have a diameter of 50-100 nm by electronmicroscopy, which is smaller than that of artificial meningococcal OMVs(diameter ˜270 nm [50]). The diameter is roughly the same as that ofartificial OMVs that have been heat-denatured (˜105 nm [50]), but thevesicles of the invention retain antigenicity whereas heat-denaturedartificial OMVs lose their antigenicity. Moreover, vesicles of theinvention (unlike MVs, OMVs and NOMVs) are substantially free fromcytoplasmic contamination.

Vesicles of the invention preferably contain no more than 20% by weightof LOS/LPS, measured relative to the total protein (i.e. there should beat least 4× more protein than LOS/LPS, by weight). The maximum LOS/LPSlevel is preferably even lower than 20% e.g. 15%, 10%, 5% or lower.

Unlike the starting culture, the vesicle-containing compositions of theinvention will generally be substantially free from whole bacteria,whether living or dead. The size of the vesicles of the invention meansthat they can readily be separated from whole bacteria by filtrationthrough a 0.22 μm filter e.g. as typically used for filtersterilisation. Thus the invention provides a process for preparingvesicles of the invention, comprising filtering the culture medium frombacteria of the invention through a filter that retards whole bacteriabut that lets the vesicles pass through e.g. a 0.22 μm filter. Althoughvesicles will pass through a standard 0.22 μm filters, these can rapidlybecome clogged by other material, and so it is preferred to performsequential steps of filter sterilisation through a series of filters ofdecreasing pore size, finishing with a standard sterilisation filter(e.g. a 0.22 μm filter). Examples of preceding filters would be thosewith pore size of 0.8 μm, 0.45 μm, etc. The filtrate can be furthertreated e.g. by ultracentrifugation.

Vesicles of the invention contain lipids and proteins. The proteincontent of meningococcal vesicles has been analysed, and substantiallyall of the proteins in the vesicles are classified as outer membraneproteins by bioinformatic analysis. Outer membrane proteins seen in thevesicles include: PilE; IgA-specific serine endopeptidase; PorA; FrpB;P1B; etc. Unlike artificial OMVs, which have previously been analysedproteomically [51], the vesicles of the invention were found to lackproteins such as MinD, FtsA and phosphoenolpyruvate synthase. Thevesicles also lack MltA.

The vesicles of the invention are advantageous when compared to vesiclesprepared by disruption of cultured bacteria because no artificialdisruption is required. Simple size-based separation can be used toseparate bacteria and vesicles, without any need for chemicaltreatments, etc. As well as being a simpler process, this avoids therisk of denaturation caused by the detergents etc. that are used duringprior art OMV preparative processes.

As mentioned above, vesicles of the invention may be similar tomicrovesicles (MVs) and ‘native OMVs’ (‘NOMVs’), which arenaturally-occurring membrane vesicles that form spontaneously duringbacterial growth and are released into culture medium. MVs can beobtained by culturing Neisseria in broth culture medium, separatingwhole cells from the broth culture medium (e.g. by filtration or bylow-speed centrifugation to pellet only the cells and not the smallervesicles) and then collecting the MVs that are present in thecell-depleted medium (e.g. by filtration, by differential precipitationor aggregation of MVs, by high-speed centrifugation to pellet the MVs).Strains for use in production of MVs can generally be selected on thebasis of the amount of MVs produced in culture. References 52 and 53describe Neisseria with high MV production.

Vesicle Combinations

The invention allows the production of immunogenic vesicles from abacterium of choice. The bacterium will typically have been generated bymutation of a chosen starting strain. Where there are multiple startingstrains of interest then the invention provides methods for preparingvesicles from each of the strains, and the different vesicles can becombined. This combination strategy is particularly useful for bacteriawhere strain-to-strain variation means that a single strain usually doesnot offer clinically-useful protection e.g. serogroup B meningococcus.

Thus the invention provides a composition comprising a mixture of n setsof vesicles of the invention, prepared from n different strains of abacterium. The value of n can be 1, 2, 3, 4, 5, etc. The differentstrains can be in the same or different serogroups. Preferred mixturesof serogroups include: A+B; A+C; A+W135; A+Y; B+C; B+W135; B+Y; C+W135;C+Y; W135+Y; A+B+C; A+B+W135; A+B+Y; A+C+W135; A+C+Y; A+W135+Y;B+C+W135; B+C+Y; C+W135+Y; A+B+C+W135; A+B+C+Y; B+C+W135+Y; andA+B+C+W135+Y.

The invention also provides a kit comprising vehicles of the inventionprepared from n different strains of a bacterium. The vesicles can bekept and stored separately in the kit until they are required to be usedtogether e.g. as an admixture, or for simultaneous separate orsequential use.

The invention also provides a process comprising: preparing n sets ofvesicles of the invention, one from each of n different strains of abacterium; and combining the n sets of vesicles. The different sets canbe combined into a kit or into an admixture.

The invention also provides the use of vesicles from a first strain of abacterium in the manufacture of a medicament for immunising a patient,wherein the medicament is administered simultaneously separately orsequentially with vesicles from a second strain of the bacterium.

The invention also the use of vesicles from a first strain of abacterium in the manufacture of a medicament for immunising a patient,wherein the patient has been pre-immunised with vesicles from a secondstrain of the bacterium.

The bacterium is preferably N.meningitidis, and is more preferably fromserogroup B. The different strains may be selected according to variouscriteria. Example criteria include: subtype and/or serosubtype [e.g.ref. 47]; immunotype; geographical origin of the strains; localprevalence of clinical strains; hypervirulent lineage e.g. one or moreof subgroups I, III and IV-1, ET-5 complex, ET-37 complex, A4 clusterand lineage 3; multilocus sequence type (MLST) [54].

Preferred criteria for selecting strains are: selection of more than onePorB serotype (class 2 or 3 OMP); selection of more than one PorAserosubtype (class 1 OMP); selection of more than one differentimmunotype (lipopolysaccharide or lipooligosaccharide); selection ofmore than one of the three different NMB1870 variants [55]. NMB1870 isseen in the vesicles of the invention, shows distinct variants, and is agood candidate antigen for vaccination [55-57]. A combination ofvesicles covering two or three different NMB1870 variants is particularadvantageous.

As well as being selected from different meningococcal strains, vesiclescan be selected from different pathogens. Thus the invention provides acomposition comprising a mixture of n sets of vesicles of the invention,prepared from n different species of bacteria. Similarly, the inventionprovides a kit comprising vesicles of the invention prepared from ndifferent species of bacteria, and provides a process comprising thestep of preparing n sets of vesicles of the invention, one from each ofn different species of bacteria.

MltA Expression

Bacteria of the invention do not possess functional MltA enzymaticactivity. Prevention of MltA protein expression can be achieved in twomain ways: removal or disruption of the endogenous mltA gene (includingits control regions) to give a MltA⁻ strain; or suppression of MltAexpression in a MltA⁺ strain. It is preferred to use a MltA⁻ strain.

MltA⁻ strains can be constructed by conventional knockout techniques.Techniques for gene knockout are well known, and meningococcus knockoutmutants of have been reported previously [e.g. refs. 25 & 58-60]. Theknockout is preferably achieved by deletion of at least a portion of thecoding region (preferably isogenic deletion), but any other suitabletechnique may be used e.g. deletion or mutation of the promoter,deletion or mutation of the start codon, etc. The bacterium may containa marker gene in place of the knocked out gene e.g. an antibioticresistance marker.

Where suppression of expression from an endogenous mltA gene is usedthen techniques such as antisense inhibition and inhibitory RNA can beused, although these techniques are more typically used in eukaryotichosts. In the resulting bacterium, mRNA encoding the knocked-out proteinwill be substantially absent and/or its translation will besubstantially inhibited (e.g. to less than 1% of the level of expressionthat would be seen in the absence of suppression).

As an alternative to knockout or suppression of expression,site-directed mutagenesis of the endogenous mltA gene can be used.Reference 61 discloses mutants of meningococcal MltA in which residuesGlu255, Glu323 and Asp362 were mutated and then tested for MltAcatalytic activity. An E255G mutant of showed a 50% reduction inactivity, and an E323G mutant showed a 70% reduction in activity.Mutagenesis of specific residues within the MltA coding region cantherefore be used as a technique to knockout the lytic transglycolaseenzymatic activity without knocking out the coding region.

Whichever technique (or combination of techniques) is chosen, theresulting bacterium will be substantially free from MltA enzymaticactivity.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising (a)vesicles of the invention and (b) a pharmaceutically acceptable carrier.The invention also provides a process for preparing such a composition,comprising the step of admixing vesicles of the invention with apharmaceutically acceptable carrier.

Typical ‘pharmaceutically acceptable carriers’ include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolised macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and lipid aggregates (such as oil dropletsor liposomes). Such carriers are well known to those of ordinary skillin the art. The vaccines may also contain diluents, such as water,saline, glycerol, etc. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, sucrose, and thelike, may be present. Sterile pyrogen-free, phosphate-bufferedphysiologic saline (e.g. pH 7.4) is a typical carrier. A thoroughdiscussion of pharmaceutically acceptable excipients is available inreference 62.

Compositions of the invention will typically be in aqueous form (i.e.solutions or suspensions) rather than in a dried form (e.g.lyophilised). Aqueous compositions are also suitable for reconstitutingother vaccines from a lyophilised form (e.g. a lyophilised Hib conjugatevaccine, a lyophilised meningococcal conjugate vaccine, etc.). Where acomposition of the invention is to be used for such extemporaneousreconstitution, the invention provides a kit, which may comprise twovials, or may comprise one ready-filled syringe and one vial, with theaqueous contents of the syringe being used to reactivate the driedcontents of the vial prior to injection.

Compositions of the invention may be presented in vials, or they may bepresented in ready-filled syringes. The syringes may be supplied with orwithout needles. Compositions may be packaged in unit dose form or inmultiple dose form. A syringe will generally include a single dose ofthe composition, whereas a vial may include a single dose or multipledoses. For multiple dose forms, therefore, vials are preferred topre-filled syringes.

Effective dosage volumes can be routinely established, but a typicalhuman dose of the composition has a volume of about 0.5 ml e.g. forintramuscular injection. The RIVM OMV-based vaccine was administered ina 0.5 ml volume [63] by intramuscular injection to the thigh or upperarm. Similar doses may be used for other delivery routes e.g. anintranasal OMV-based vaccine for atomisation may have a volume of about100 μl or about 130 μl per spray [64], with four sprays administered togive a total dose of about 0.5 ml.

The pH of the composition is preferably between 6 and 8, and morepreferably between 6.5 and 7.5 (e.g. about 7 or about 7.4). The pH ofthe RIVM OMV-based vaccine is 7.4 [65], and a pH <8 (preferably <7.5) ispreferred for compositions of the invention. Stable pH may be maintainedby the use of a buffer e.g. a Tris buffer, a phosphate buffer, or ahistidine buffer. Compositions of the invention will generally include abuffer. If a composition comprises an aluminium hydroxide salt, it ispreferred to use a histidine buffer [66] e.g. at between 1-10 mM,preferably about 5 mM. The RIVM OMV-based vaccine maintains pH by usinga 10 mM Tris/HCl buffer. The composition may be sterile and/orpyrogen-free. Compositions of the invention may be isotonic with respectto humans.

Compositions of the invention are immunogenic, and are more preferablyvaccine compositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic. Immunogenic compositionsused as vaccines comprise an immunologically effective amount ofantigen(s), as well as any other components, as needed. By‘immunologically effective amount’, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, age, the taxonomic group of individual to be treated (e.g.non-human primate, primate, etc.). the capacity of the individual'simmune system to synthesise antibodies, the degree of protectiondesired, the formulation of the vaccine, the treating doctor'sassessment of the medical situation, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials. The antigen content ofcompositions of the invention will generally be expressed in terms ofthe amount of protein per dose. A dose of about 0.9 mg protein per ml istypical for OMV-based intranasal vaccines [64]. The MeNZB™ OMV-basedvaccine contains between 25 and 200 μg of protein per millilitre e.g.between 45 and 90 μg/ml, or 50±10 μg/ml. Compositions of the inventionpreferably include less than 100 μg/ml of OMV per strain of bacterium.

Meningococci affect various areas of the body and so the compositions ofthe invention may be prepared in various forms. For example, thecompositions may be prepared as injectables, either as liquid solutionsor suspensions. The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as spray,drops, gel or powder [e.g. refs. 67 & 68].

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format. Antimicrobials such as thiomersaland 2-phenoxyethanol are commonly found in vaccines, but it is preferredto use either a mercury-free preservative or no preservative at all.

Compositions of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical. The concentration of sodium chloride is preferably greater than7.5 mg/ml.

Compositions of the invention will generally be administered inconjunction with other immunoregulatory agents. In particular,compositions will usually include one or more adjuvants, and theinvention provides a process for preparing a composition of theinvention, comprising the step of admixing vesicles of the inventionwith an adjuvant e.g. in a pharmaceutically acceptable carrier. Suitableadjuvants include, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 69], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt [70].

A typical aluminium phosphate adjuvant is amorphous aluminiumhydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92, includedat 0.6 mg Al³⁺/ml. Adsorption with a low dose of aluminium phosphate maybe used e.g. between 50 and 100 μg Al³⁺ per conjugate per dose. Where analuminium phosphate it used and it is desired not to adsorb an antigento the adjuvant, this is favoured by including free phosphate ions insolution (e.g. by the use of a phosphate buffer).

The RIVM vaccine was tested with adsorption to either an aluminiumphosphate or an aluminium hydroxide adjuvant, and the aluminiumphosphate adjuvant was found to give superior results [65]. The MeNZB™,MenBvac™ abd VA-MENINGOC-BC™ products all include an aluminium hydroxideadjuvant.

A typical dose of aluminium adjuvant is about 3.3 mg/ml (expressed asAl³⁺ concentration).

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MP59 [Chapter 10 of ref. 69;see also ref. 71] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

C. Saponin Formulations [Chapter 22 of Ref. 69]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla panicitlata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified factions using these techniques have been identified, includingQS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin isQS21. A method of production of QS21 is disclosed in ref. 72. Saponinformulations may also comprise a sterol, such as cholesterol [73].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexs (ISCOMs) [chapter 23 of ref.69]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA and QHC. ISCOMs are further described in refs. 73-75. Optionally,the ISCOMS may be devoid of extra detergent [76].

A review of the development of saponin based adjuvants can be found inrefs. 77 & 78.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 79-84. Virosomes are discussed further in, for example, ref. 85

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 86. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [86]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [87,88].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 89 & 90.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 91, 92 and 93 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 94-99.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [100]. The CpG sequence may be specific for inducing a Thl immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 101-103. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 100 & 104-106.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E.coli (E.coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 107 and as parenteraladjuvants in ref. 108. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant in a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivaties thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 109-116. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 117, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [118], etc.) [119], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [120] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [121].

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref. 69)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 122-124.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [125]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [126] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [127]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 128 and 129.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 130 and 131.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [132]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL [133]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) [134]; (5) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions [135]; (6) SAF,containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L12I,and thr-MDP, either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion. (7) Ribi™ adjuvant system(RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and oneor more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); and (8) one or more mineralsalts (such as an aluminum salt)+a non-toxic derivative of LPS (such as3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 69.

The use of aluminium salt adjuvants is particularly preferred, andantigens are generally adsorbed to such salts. It is possible incompositions of the invention to adsorb some antigens to an aluminiumhydroxide but to have other antigens in association with an aluminiumphosphate. In general, however, it is preferred to use only a singlesalt e.g. a hydroxide or a phosphate, but not both. Not all vesiclesneed to be adsorbed i.e. some or all can be free in solution.

Methods of Treatment

The invention also provides a method for raising an immune response in amammal, comprising administering a pharmaceutical composition of theinvention to the mammal. The immune response is preferably protectiveand preferably involves antibodies. The method may raise a boosterresponsein a patient that has already been primed againstN.meningitidis. Subcutaneous and intranasal prime/boost regimes for OMVsare disclosed in ref. 136.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc.

The invention also provides vesicles of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of vesicles of the invention in themanufacture of a medicament for raising an immune response in a mammal.

The invention also the use of vesicles of the invention in themanufacture of a medicament for immunising a patient, wherein thepatient has been pre-immunised with at least one of the following:diphtheria toxoid; tetanus toxoid; acellular or cellular pertussisantigens; a conjugated Hib capsular saccharide; hepatitis B virussurface antigen; a conjugated meningococcal capsular saccharide; and/ora conjugated pneumococcal capsular saccharide.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by N.meningitidis e.g. bacterial (or, morespecifically, meningococcal) meningitis, of septicemia.

One way of checking efficacy of therapeutic treatment involvesmonitoring Neisserial infection after administration of the compositionof the invention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the vesicles' antigensafter administration of the composition. Immunogenicity of compositionsof the invention can be determined by administering them to testsubjects (e.g. children 12-16 months age, or animal models [137]) andthen determining standard parameters including serum bactericidalantibodies (SBA) and ELISA titres (GMT). These immune responses willgenerally be determined around 4 weeks after administration of thecomposition, and compared to values determined before administration ofthe composition. A SBA increase of at least 4-fold or 8-fold ispreferred. Where more than one dose of the composition is administered,more than one post-administration determination may be made.

Preferred compositions of the invention can confer an antibody titre ina patient that is superior to the criterion for seroprotection for anacceptable percentage of human subjects. Antigens with an associatedantibody titre above which a host is considered to be seroconvertedagainst the antigen are well known, and such titres are published byorganisations such as WHO. Preferably more than 80% of a statisticallysignificant sample of subjects is seroconverted, more preferably morethan 90%, still more preferably more than 93% and most preferably96-100%.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined. The OMV-based RIVM vaccine was tested using a3- or 4-dose primary schedule, with vaccination at 0, 2 & 8 or 0, 1, 2 &8 months. MeNZB™ is administered as three doses at six week intervals.These schedules can be used according to the invention. The vesiclepreparations given at each dose stage can be the same or different.

In methods of the invention, where a first dose is given at time zerothen a second and a third dose may be given over the next two months,and a fourth dose may be given between 11 and 13 months after time zero.The first, second and third doses may comprise vesicles with the sameserosubtype as each other, and the fourth dose may comprises vesicleswith a different serosubtype from the first three doses. The fourth dosemay contain vesicles only with a different serosubtype from the firstthree doses, or it may contain two types of vesicle, one with adifferent serosubtype from the first three doses and one with the samesubtype. The first, second and third doses are preferably given atintervals of between 6 and 8 weeks. The fourth dose is preferably givenabout 1 year after time zero. The patient preferably receives the samequantity of vaccine at each of the four doses.

As described above, the invention may involve administration of vesiclesfrom more than one subtype and/or serosubtype of N.meningitidis [e.g.ref. 47], either separately or in admixture.

The invention may be used to elicit systemic and/or mucosal immunity.

In general, compositions of the invention are able to induce serumbactericidal antibody responses after being administered to a subject.These responses are conveniently measured in mice and are a standardindicator of vaccine efficacy [e.g. see end-note 14 of reference 196].Serum bactericidal activity (SBA) measures bacterial killing mediated bycomplement, and can be assayed using human or baby rabbit complement.WHO standards require a vaccine to induce at least a 4-fold rise in SBAin more than 90% of recipients. MeNZB™ elicits a 4-fold rise in SBA 4-6weeks after administration of the third dose.

Rather than offering narrow protection, compositions of the inventioncan induce bactericidal antibody responses against more than onehypervirulent lineage of serogroup B. In particular, they can preferablyinduce bactericidal responses against two or three of the followingthree hypervirulent lineages: (i) cluster A4; (ii) ET5 complex; and(iii) lineage 3. They may additionally induce bactericidal antibodyresponses against one or more of hypervirulent lineages subgroup I,subgroup III, subgroup IV-1 or ET-37 complex, and against other lineagese.g. hyperinvasive lineages. This does not necessarily mean that thecomposition can induce bactericidal antibodies against each and everystrain of serogroup B meningococcus within these hypervirulent lineagese.g. rather, for any given group of four of more strains of serogroup Bmeningococcus within a particular hypervirulent linage, the antibodiesinduced by the composition are bactericidal against at least 50% (e.g.60%, 70%, 80%, 90% or more) of the group. Preferred groups of strainswill include strains isolated in at least four of the followingcountries: GB, AU, CA, NO, IT, US, NZ, NL, BR, and CU. The serumpreferably has a bactericidal titre of at least 1024 (e.g. 2¹⁰, 2¹¹,2¹², 2¹³, 2¹⁴, 2¹⁵, 2¹⁶, 2¹⁷, 2¹⁸ or higher, preferably at least 2³⁴)e.g. the serum is able to kill at least 50% of test bacteria of aparticular strain when diluted 1/1024, as described in reference 196.

Preferred compositions can induce bactericidal responses against thefollowing strains of serogroup B meningococcus: (i) from cluster A4,strain 961-5945 (B:2b:P1.21,16) and/or strain G2136 (B:−); (ii) fromET-5 complex, strain MC58 (B:15:P1.7,16b) and/or strain 44/76(B:15:P1.7,16); (iii) from lineage 3, strain 394/98 (B:4:P1.4) and/orstrain BZ198 (B:NT:−). More preferred compositions can inducebactericidal responses against strains 961-5945, 44/76 and 394/98.

Strains 961-5945 and G2136 are both Neisseria MLST reference strains[ids 638 & 1002 in ref. 138]. Strain MC58 is widely available (e.g. ATCCBAA-335) and was the strain sequenced in reference 32. Strain 44/76 hasbeen widely used and characterised (e.g. ref. 139) and is one of theNeisseria MLST reference strains [id 237 in ref. 138; row 32 of Table 2in ref. 33]. Strain 394/98 was originally isolated in New Zealand in1998, and there have been several published studies using this strain,(e.g. refs. 140 & 141). Strain BZ198 is another MLST reference strain[id 409 in ref. 138; row 41 of Table 2 in ref. 33].

Further Antigenic Components

As well as containing antigenic vesicles of the invention, compositionsof the invention may include further non-vesicular antigens. Forexample, the composition may comprise one or more of the followingfurther antigens

-   -   saccharide antigen from N.meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 142 from        serogroup C or the oligosaccharides of ref. 143. The        VA-MENINGOC-BC™ product contains serogroup C polysaccharide.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. refs.        144-146; chapters 22 & 23 of ref. 153].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 147, 148; chapter 15 of ref. 153].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 148,149; chapter 16 of ref. 153].    -   an antigen from hepatitis C virus [e.g. 150].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from        B.pertussis, optionally also in combination with pertactin        and/or agglutinogens 2 and 3 [e.g. refs. 151 & 152; chapter 22        of ref. 153].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        13 of ref. 153].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 27 of        ref. 153].    -   a saccharide antigen from Haemophilus influenzae B [e.g. chapter        14 of ref. 153]    -   an antigen from N.gonorrhoeae [e.g. ref. 154].    -   an antigen from Chlamydia pneumoniae [e.g. 155-161].    -   an antigen from Chlamydia trachomatis [e.g. 162].    -   an antigen from Porphyromonas gingivalis [e.g. 163].    -   polio antigen(s) [e.g. 164, 165; chapter 24 of ref. 153] such as        IPV.    -   rabies antigen(s) [e.g. 166] such as lyophilised inactivated        virus [e.g. 167, RabAvert™].    -   measles, mumps and/or rubella antigens [e.g. chapters 19, 20 and        26 of ref. 153].    -   influenza antigen(s) [e.g. chapters 17 & 18 of ref. 153], such        as the haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. 168].    -   a protein antigen from Streptococcus agalactiae (group B        streptococcus) [e.g. 169, 170].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 170, 171, 172].

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier in order to enhance immunogenicity. Conjugationof H.influenzae B, meningococcal and pneumococcal saccharide antigens iswell known.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or generic means[152]).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens. DTP combinations are thus preferred.

Saccharide antigens are preferably in the form of conjugates. Preferredcarrier proteins for conjugates are bacterial toxins or toxoids, such asdiphtheria toxoid or tetanus toxoid. The CRM197 mutant of diphtheriatoxin [173-175] is a particularly preferred carrier for, as is adiphtheria toxoid. Other suitable carrier proteins include theN.meningitidis outer membrane protein [176], synthetic peptides[177,178], heat shock proteins [179,180], pertussis proteins [181,182],cytokines [183], lymphokines [183], hormones [183], growth factors[183], artificial proteins comprising multiple human CD4⁺ T cellepitopes from various pathogen-derived antigens [184] such as N19,protein D from H.influenzae [185,186], pneumococcal surface protein PspA[187], pneumolysin [188], iron-uptake proteins [189], toxin A or B fromC.difficile [190], etc.

Preferred compositions include meningococcal Vesicles as describedabove, plus a conjugated capsular saccharide from one or more (i.e. 1,2, 3 or 4) of meningococcal serogroups A, C, W135 and Y. Where theVesicles are from serogroup B then this approach allows the followingserogroups to be covered: B+A; B+C; B+W135; B+Y; B+C+W135; B+C+Y;B+W135+Y; B+A+C+W135; B+A+C+Y; B+A+W135+Y; B+C+W135+Y; and B+A+C+W135+Y.Two preferred combinations use serogroup B Vesicles plus conjugateantigens from either serogroups A+W135+Y or serogroups A+C+W135+Y. Ingeneral, it is possible to cover all five of serogroups A, B, C, W135and Y by choosing Vesicles for x serogroup(s) and conjugated saccharidesfor the remaining 5-x serogroups.

Specific meningococcal protein antigens (preferably from serogroup B)may also be added to supplement the vesicle compositions. In particular,a protein antigen such as disclosed in refs. 41 & 191 to 199 may beadded. A small number of defined antigens may be added (a mixture of 10or fewer (e.g. 9, 8, 7, 6, 5, 4, 3, 2) purified antigens). Preferredadditional immunogenic polypeptides for use with the invention are thosedisclosed in reference 199: (1) a ‘NadA’ protein; (2) a ‘741’ protein;(3) a ‘936’ protein; (4) a ‘953’ protein; and (5) a ‘287’ protein. Otherpossible supplementing meningococcal antigens include transferrinbinding proteins (e.g. TbpA and TbpB) and/or Cu,Zn-superoxide dismutase[18]. Other possible supplementing meningococcal antigens include ORF40(also known as ‘Hsf’ or ‘NhhA’ [200,201]), LetP [202] and ExbB [202].Other possible supplementing meningococcal antigens include proteinscomprising one of the following amino acid sequences: SEQ ID NO:650 fromref. 191; SEQ ID NO:878 from ref. 191; SEQ ID NO:884 from ref. 191; SEQID NO:4 from ref. 192; SEQ ID NO:598 from ref. 193; SEQ ID NO:818 fromref. 193; SEQ ID NO:864 from ref. 193; SEQ ID NO:866 from ref. 193; SEQID NO:1196 from ref. 193; SEQ ID NO:1272 from ref. 193; SEQ ID NO:1274from ref. 193; SEQ ID NO:1640 from ref. 193; SEQ ID NO:1788 from ref.193; SEQ ID NO:2288 from ref. 193; SEQ ID NO:2466 from ref. 193; SEQ IDNO:2554 from ref. 193; SEQ ID NO:2576 from ref. 193; SEQ ID NO:2606 fromref. 193; SEQ ID NO:2608 from ref. 193; SEQ ID NO:2616 from ref. 193;SEQ ID NO:2668 from ref. 193; SEQ ID NO:2780 from ref. 193; SEQ IDNO:2932 from ref. 193; SEQ ID NO:2958 from ref. 193; SEQ ID NO:2970 fromref. 193; SEQ ID NO:2988 from ref. 193, or a polypeptide comprising anamino acid sequence which: (a) has 50% or more identity (e.g. 60%, 70%,80%, 90%, 95%, 99% or more) to said sequences; and/or (b) comprises afragment of at least n consecutive amino acids from said sequences,wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments for(b) comprise an epitope from the relevant sequence. More than one (e.g.2, 3, 4, 5, 6) of these polypeptides may be included. The meningococcalantigens transferrin binding protein and/or Hsf protein may also beadded [203].

Supplementation of the OMVs by defined meningococcal antigens in thisway is particularly useful where the OMVs are from a serosubtype P1.7b,4meningococcus or a serosubtype P1.7,16 meningococcus. Supplementation ofa mixture of OMVs from both these serosubtypes is preferred.

It is also possible to add vesicles that are not vesicles of theinvention e.g. OMVs, MVs, NOMVs, etc. that are prepared by methods otherthan those of the invention (e.g. prepared by methods involvingdisruption of bacterial membranes, as disclosed in the prior art).

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using protein antigens in the composition of theinvention, nucleic acid encoding the antigen may be used. Proteincomponents of the compositions of the invention may thus be replaced bynucleic acid (preferably DNA e.g. in the form of a plasmid) that encodesthe protein.

New Meningococcal Proteins

The genome sequence of serogroup B meningococcus is reported inreference 32. The initial annotation of the genome has not been acceptedfor all of the >2000 genes e.g. the start codon on NMB1870 hassubsequently been re-assigned [41,55]. The inventors have found that thestart codons for NMB0928, NMB0109 and NMB1057 should also bere-assigned:

-   -   The original sequence of NMB0928 is shown in FIG. 6 (SEQ ID NO:        3). The inventors believe that the true start codon for NMB0928        in the ATG encoding the methionine at residue 24 of FIG. 6. With        the new start codon (SEQ ID NO: 6), NMB0928 presents a typical        signature of a surface-exposed protein, characterised by a        signal peptide with a lipo-box motif (underlined).    -   The original sequence of NMB0109 is shown in FIG. 7 (SEQ ID NO:        4). The inventors believe that the true start codon for NMB0109        is the ATG encoding the Met at residue 39 of FIG. 7. (SEQ ID NO:        7)    -   The original sequence of NMB1057 is shown in FIG. 8 (SEQ ID NO:        5). The inventors believe that the true start codon for NMB1057        is the GTG encoding the Val at residue 14 of FIG. 8. (SEQ ID NO:        8)

Thus the invention provides a polypeptide comprising: (a) the amino acidsequence of SEQ ID NO:6; (b) an amino acid sequence having at least 50%(e.g. 60%, 70%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)sequence identity to SEQ ID NO:6, and/or comprising an amino acidsequence consisting of a fragment of at least 7 (e.g. 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250)contiguous amino acids from SEQ ID NO:6. Preferred polypeptides have aN-terminus cysteine residue, preferably corresponding to Cys-19 of SEQID NO:6, and the N-terminus cysteine is preferably lipidated. Preferredpolypeptides do not include the amino acid sequence MTHIKPVIAALALIGLAA(SEQ ID NO: 9) within 30 amino acids of their N-terminus.

The invention also provides a polypeptide comprising: (a) the amino acidsequence of SEQ ID NO:7; (b) an amino acid sequence having at least 50%(e.g. 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)sequence identity to SEQ ID NO:7, and/or comprising an amino acidsequence consisting of a fragment of at least 7 (e.g. 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250)contiguous amino acids from SEQ ID NO:7. Preferred polypeptides do notinclude the amino acid sequence MLKCGTFFITRHIPRGCRRFFQPNQARQTEIYQIRGTV(SEQ ID NO: 10) within 20 amino acids of their N-terminus.

The invention also provides a polypeptide comprising: (a) the amino acidsequence of SEQ ID NO:8; (b) an amino acid sequence having at least 50%(e.g. 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)sequence identity to SEQ ID NO:8, and/or comprising an amino acidsequence consisting of a fragment of at least 7 (e.g. 8, 9, 10, 11 ,12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250)contiguous amino acids from SEQ ID NO:8. Preferred polypeptides have aN-terminus cysteine residue, preferably corresponding to Cys-Gln of SEQID NO:8, and the N-terminus cysteine is preferably lipidated. Otherpreferred polypeptides do not include the amino acid sequence MPCMNHQSNS(SEQ ID NO: 11) within 20 amino acids of their N-terminus.

Polypeptides can be prepared by various means e.g. by chemical synthesis(at least in part), by digesting longer polypeptides using proteases, bytranslation from RNA, by purification from cell culture (e.g. fromrecombinant expression or from N.meningitidis culture), etc.Heterologous expression in an E.coli host is a preferred expressionroute.

Polypeptides of the invention may be attached or immobilised to a solidsupport. Polypeptides of the invention may comprise a detectable labele.g. a radioactive label, a fluorescent label, or a biotin label. Thisis particularly useful in immunoassay techniques.

Polypeptides can take various forma (e.g. native, fusions, glycosylated,non-glycosylated, lipidated, disulfide bridges, etc.). Polypeptides arepreferably meningococcal polypeptides.

Polypeptides are preferably prepared in substantially pure orsubstantially isolated form (i.e. substantially free from otherNeisserial or host cell polypeptides) or substantially isolated form. Ingeneral, the polypeptides are provided in a non-naturally occurringenvironment e.g. they are separated from their naturally-occurringenvironment. In certain embodiments, the subject polypeptide is presentin a composition that is enriched for the polypeptide as compared to acontrol. As such, purified polypeptide is provided, whereby purified ismeant that the polypeptide is present in a composition that issubstantially free of other expressed polypeptides, where bysubstantially free is meant that less than 50%, usually less than 30%and more usually less than 10% of the composition is made up of otherexpressed polypeptides.

The term “polypeptide” refers to amino acid polymers of any length. Thepolymer may be linear or branched, it may comprise modified amino acids,and it may be interrupted by non-amino acids. The terms also encompassan amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art.Polypeptides can occur as single chains or associated chains.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 ofreference 204. A preferred alignment is determined by the Smith-Watermanhomology search algorithm using an affine gap search with a gap openpenalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is well known and is disclosedin reference 205.

References to ‘NMB’ proteins with a four digit number refers toB thestandard nomenclature of reference 32, assigned on the basis of a genomesequence of a prototypic strain of serogroup B meningococcus. The publicsequence databases include these NMB sequences- For any givenmeningococcus, the skilled person can readily and unambiguously find thegene corresponding to a NMBnnnn sequence by using the existing sequencefrom the database and/or the genetic environment of the NMBnnnn ORF inthe prototype strain e.g. to design primers, probes, etc.

The terms ‘GNA33’, ‘NMB0033’ and ‘mltA’ can be used interchangeably whenreferring to meningococcus.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows 2D-PAGE of vesicles of the invention.

FIGS. 2A-B show the gel filtration outputs with standard proteins (FIG.2B) and with the centrifugation pellet (FIG. 2A) of the culturesupernatant of the ΔmltA strain. They y-axis on both FIG. 2A and FIG. 2Bshows absorbance at 280 nm.

FIGS. 3A-B show electron microscopy of vesicles of the invention. FIG.3A is lower resolution electron microscopy (the bar shows 100 nm). FIG.3B is higher resolution electron microscopy (the bar shows 50 nm).

FIGS. 4A-F shows western blot analysis of vesicles of the invention withsix different antibodies. FIG. 4A shows mouse serum raised against OMVsprepared from the NZ strain by deoxycholate extraction. FIG. 4B showsmouse serum raised against ΔGNA33 knockout mutants. FIG. 4C shows mouseanti-PorA_(P1.4) monoclonal. FIG. 4D shows mouse anti-NMB2132 serum.FIG. 4E shows mouse anti-NMB1030 serum. FIG. 4F shows mouse anti-NMB1870serum.

FIG. 5 compares the proteins released into culture supernatants bywild-type or ΔGNA33 bacteria. Lane 1: Molecular weight markers; lane 2:culture medium control; lane 3: 20 μg proteins collected by high speedcentrifugation of ΔGNA33 culture medium at OD_(620 nm)=0.5,corresponding to 6.5 ml of culture medium; lane 4: proteins collected byhigh speed centrifugation from 6.5 ml of wild-type MC58 culture mediumat OD_(620 nm)=0.5.

FIG. 6 shows SDS-PAGE of a wild-type MC58 total extract (lanes 2 and 4)and of vesicles released by ΔGNA33 knockout mutant (lanes 3 and 5).Lanes 2 and 3 are proteins not denatured at 95° C. prior to SDS-PAGE;lanes 4 and 5 were denatured at 95° C.

FIGS. 7A-B show 1D SDS-PAGE (FIG. 7A) and 2D SDS-PAGE (FIG. 7B) ofvesicles prepared from strain 394/98. In FIG. 7B the horizontal axisruns from pI 3 to 10 and the vertical axis runs from 10 to 200 kDa.

FIGS. 8A-B show 1D SDS-PAGE of vesicles prepared from tolR ExPECknockout strains. FIG. 8A shows two preparations of E. coli CFI073. FIG.8B shows the E. coli strain preparations: DH5a, 536, and IHE3034.

FIGS. 9A-D show 1D SDS-PAGE (FIGS. 9A and 9C) and 2D SDS-PAGE (FIGS. 9Band 9D) of vesicles from ΔmltA knockout meningococci. FIGS. 9A and 9Bare strain MC58. FIGS. 9C and 9D are strain NZ98/254.

FIGS. 10A-B show 1D SDS-PAGE and 2D SDS-PAGE (FIG. 10B) of vesicles fromΔmltA knockout meningococci.

The amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ IDNO: 2) are the membrane-bound lync nurein transglyosclyase A (mlta) fromthe genome sequence of strain MC59 of serogroup B Neisseriameningitidis, taken from GenBank accession AAF405041.1 [32]. SEQ ID NO:3 is NMB0928, SEQ ID NO: 4 is NMB0109, SEQ ID NO: 5 is NMB 1057, SEQ IDNO: 6 is NMB0928_(new), and SEQ ID NO: 8 is NMB1057_(new), which bothhave shifted start codons.

MODES FOR CARRYING OUT THE INVENTION

Preparation of Meningococcal mltA Knockout Strain

A meningococcal strain was prepared in which the mltA gene is replacedby allelic exchange with an antibiotic cassette.

N.meningitidis strain MC58 was transformed with plasmid pBSUDGNA33ERM.This plasmid contains upstream and downstream flanking regions forallelic exchange, a truncated mltA gene, and the ermC gene (encodingerythromycin resistance). The upstream flanking region (including thestart codon) from position −867 to +75 and the downstream flankingregion (including the stop codon) from position +1268 to +1744 wereamplified from MC58 by using the primers U33FOR, U33REV, D33FTOR andD33REV [25]. Fragments were cloned into pBluescript™ and transformedinto E.coli DH5 by using standard techniques. Once all subcloning wascomplete, naturally competent Neisseria strain MC58 was transformed byselecting a few colonies grown overnight on GC agar plates and mixingthem with 20 μl 10 mM Tris-HCl (pH 6.5) containing 1 μg plasmid DNA. Themixture was spotted onto a chocolate agar plate, incubated for 6 h at37° C. with 5% CO₂, and then diluted in phosphate buffered-saline (PBS)and spread on GC agar plates containing 7 μg/ml erythromycin. Allelicexchange with the chromosomal mltA gene was verified by PCR, and lack ofMltA expression was confirmed by Western blot analysis.

As reported in reference 25, the mltA knockout strain does not have thecorrect topological organisation of the cellular membrane, has abnormalcell separation, abnormal cell morphology, undivided septa, doublesepta, cell clustering, sharing of outer membranes and reducedvirulence. Reference 25 also reports that the knockout strain releasesvarious membrane proteins into the culture supernatant, including thePorA, PIB, class 4 and class 5 outer membrane proteins.

A mltA knockout was also made from New Zealand strain 394/98 (lin3;B:4:P1.4), which is the strain from which the MeNZB™ product isproduced.

Analysis of Released Proteins

The ΔmltA strain was grown in GC culture medium in a humidifiedatmosphere containing 5% CO₂ until OD_(600 nm) 0.5. Bacteria werecollected by 10 minutes of centrifugation at 3500× g. The supernatant(i.e. culture medium) was filtered through a 0.22 μm pore size filter(Millipore), and the cell-free filtrate was subjected to high-speedcentrifugation (200,000× g, 90 min). This centrifugation resulted information of a pellet, with about 8-12 mg protein per litre of culturemedium. No such pellet was seen if wild-type MC58 bacteria were treatedin the same way, and so the pellet formation is a result of the mltAknockout. The pellet was washed twice with PBS (centrifugation200,000×g, 30 min) for further analysis.

In a first analysis, material from the pellet was re-suspended in PBSand applied to a Superdex 200 PC3.2/30 gel filtration column, run on aSMART system (Amersham Biosciences) that had been equilibrated in PBS.The flow rate was 40 μl/min, and eluate was monitored at 280 nm. Thecolumn was calibrated with 20 μg Bleu dextran (2,000 kDa), 10 μgferritine (440 kDa), 140 μg bovine serum albumin (65 kDa) and 200 μgribonuclease A (15 kDa). As shown in FIG. 3, most of the proteins elutedin a major peak corresponding to a molecular weight substantially higherthan 2,000 kDa. This result suggests that the various proteins areassociated.

In a second analysis, the material present in the high molecular weightpeak was subjected to negative staining electron microscopy. Thisanalysis revealed the presence of well-organised membrane vesicles witha diameter of about 50-100 nm (FIG. 4).

These experiments suggest that deletion of the mltA gene perturbs thenormal assembly of the bacterial membrane, and that this results in thespontaneous release into the culture supernatant of membrane structureswhich assemble in spherical, homogeneous vesicles.

FIG. 12 shows SDS-PAGE analysis of culture media after growth ofwild-type or ΔGNA33 bacteria, and shows the different protein releasecharacteristics.

Analysis of Vesicles

The ΔmltA-derived vesicles were compared to meningococcal vesiclesprepared by the ‘normal’ detergent extraction method.

Meningococcal strains MC58, NZ394/98 and NZ98/254, and their respectiveisogenic ΔmltA mutants, were grown in 20 ml or 200 ml GC culture mediumin humidified atmosphere containing 5% CO₂ until OD_(620 nm) 0.5.Bacteria were collected by 10-minute centrifugation at 3500 g. Vesicles(‘DOMVs’) were prepared from the wild-type bacteria by detergentextraction as described in reference 206. Vesicles of the invention(‘mOMVs’) were prepared from knockout strains by filtration through a0.22 μm pore size filter, followed by high-speed centrifugation (200,000g, 90 min) of the filtrates, washing of the vesicle-containing pellets(centrifugation 200,000 g, 30 min) twice with phosphate buffer saline,(PBS), and then re-suspension with PBS.

Both the mOMVs and the DOMVs were analysed by denaturingmono-dimensional electrophoresis. Briefly, 20 μg of vesicle proteinswere resolved by SDS-PAGE and visualised by Coomassie Blue staining of12.5% gels. Denaturing (2% SDS) and semi-denaturing (0.2% SDS, nodithiothreitol, no heating) conditions were used mono-dimensionalelectrophoresis. The amount of protein (20 μg) was determined by DCprotein assay (Bio-Rad), using bovine serum albumin as a standardprotein.

The vesicles were denatured for 3 minutes at 95° C. in SDS-PAGE samplebuffer containing 2% SDS. 20 μg of protein were then loaded onto 12.5%acrylamide gels, which were stained with Coomassie Blue R-250.2-dimensional electrophoresis was also performed on 200 μg of proteinbrought to a final volume of 125UI with re-swelling buffer containing 7Murea, 2M thiourea, 2% (w/v)(3-((3-cholamidopropyl)dimethylammonio)-1-propane-sulfonate), 65 mMdithiothreitol, 2% (w/v) amidosulfobetain-14, 2 mM tributylphosphine, 20mM Tris, and 2% (v/v) carrier ampholyte. Proteins were adsorbedovernight onto Immobiline DryStrips (7 cm; pH-gradient 3-10 non linear).Proteins were then 2D-separated. The first dimension was run using aIPGphor Isoelectric Focusing Unit, applying sequentially 150 V for 35min., 500 V for 35 min., 1,000 V for 30 min, 2,600 V for 10 min., 3,500V for 15 min., 4,200 V for 15 min., and finally 5,000 V to reach 12 kVh.For the second dimension, the strips were equilibrated and proteins wereseparated on linear 9-16.5% polyacrylamide gels (1.5-mm thick, 4×7 cm).Gels were again stained with Coomassie Brilliant Blue G-250. 266 proteinspots could be seen after Colloidal Coomassie Blue staining (FIG. 2).

The 1D and 2D gels were then subjected to in-gel protein digestion andsample preparation for mass spectrometry analysis. Protein spots wereexcised from the gels, washed with 100 mM ammoniumbicarbonate/acetonitrile 50/50 (V/V), and dried using a SpeedVaccentrifuge. Dried spots were digested 2 hours at 37° C. in 12 μl of0.012 μg/μl sequencing grade trypsin (Promega) in 50 mM ammoniumbicarbonate, 5 mM. After digestion, 5 μl of 0.1% trifluoacetic acid wasadded, and the peptides were desalted and concentrated with ZIP-TIPs(C18, Millipore). Sample were eluted with 2 μl of 5 g/l2,5-dihydroxybenzoic acid in 50% acetonitrile/0.1% trifluoroacetic acidonto the mass spectrometer Auchorchip 384 (400 μm, Bruker, Bremen,Germany) and allowed to air dry at room temperature. MALDI-TOF spectrawere acquired on a Bruker Biflex III MALDI-TOF equipped with a 337 nm N₂laser and a SCOUT 384 multiprobe ion source set in a positive-ionreflector mode. The acceleration and reflector voltages were set at 19kV and 20 kV, respectively. Typically, each spectrum was determined byaveraging 100 laser shots. Spectra were externally calibrated using acombination of four standard peptides, angiotensin II (1,046.54 Da),substance P (1,347.74 Da), Bombensin (1,619.82 Da) and ACTH18-39 Cliphuman (2,465.20 Da), spotted onto adjacent position to the samples.Protein identification was carried out by both automatic and manualcomparison of experimentally-generated monoisotopic values of peptidesin the mass range of 700-3000 Da with computer-generated fingerprintsusing the Mascot software.

Results from the MC58 ΔmltA mutant are shown in FIG. 18. From the 20excised bands on just the 1D gel, 25 unique proteins were identified, 24(96%) of which were predicted to be outer-membrane proteins by the PSORTalgorithm (Table 1 below). 170 protein spots on the 2D gel,corresponding to 51 unique proteins, were unambiguously identified byMALDI-TOF (Table 1). 44/51 identified proteins have been assigned to theouter membrane compartment by the genome annotation [32]. The 7remaining proteins were analysed for possible errors in the originalannotation. Four proteins (the hypothetical proteins NMB1870, NMB0928and NMB0109, and the glutamyltranspeptidase NMB1057) could be classifiedas outer membrane proteins using different start codons from those inref. 32 e.g. for NMB1870, using the start codon assigned in reference55.

The combined 1D and 2D electrophoresis experiments identified a total of65 proteins in the MC58 ΔmltA mutant-derived vesicles. Of these, 6proteins were identified in both 1D and 2D gels, whereas 14 and 45 werespecific for the 1D and 2D gels, respectively (Table 1). Moreover, 61out of the 65 identified proteins were predicted as membrane-associatedproteins by current algorithms, indicating that the ΔmltA vesicles(mOMVs) are mostly, and possible exclusively, constituted by membraneproteins.

The ΔmltA knockout of strain NZ394/98 was similarly subjected to 1D and2D SDS-PAGE (FIGS. 14 & 15). Table 2 shows 66 proteins that wereidentified in one or both of the gels, together with the predictedlocation of the proteins. Again, most of the proteins were predicted asmembrane-associated. The 47 proteins common to Tables 1 and 2 are shownin Table 3.

Results from the NZ98/254 ΔmltA mutant are shown in FIG. 19. 66 proteinswere identified from these two gels, 57 of which were assigned to theouter membrane compartment. Again, therefore, the mOMVs are highlyenriched in outer membrane proteins. 46 of the 57 proteins had also beenidentified in the MC58-derived mOMVs.

For comparison, FIG. 20 shows the results from NZ98/254 DOMVs. Proteomicanalysis revealed 138 proteins, only 44 of which were assigned to theouter membrane compartment. The remaining 94 proteins belonged to thecytoplasmic and inner membrane compartments. Of these 44 membraneproteins, 32 were also found in the 57 outer membrane proteins found inthe mOMVs from the isogenic strain.

While mOMVs are largely constituted by outer membrane proteins,therefore, about 70% of DOMV proteins are either cytoplasmic or innermembrane proteins. DOMVs differ from mOMVs not only for the proportionof cytoplasmic proteins but also for the different profile of theirouter membrane proteins. Of the 44 outer membrane proteins seen inDOMVs, only 32 were also seen in mOMVs.

19 proteins seen in mOMVs from both MC58 and NZ98/254, but not in theDOMVs from NZ98/254, are listed in Table 4 below.

A total cell extract of bacteria was prepared as follows: Bacterialcells were washed with PBS, and the bacterial pellet was resuspended in8 ml of 50 mM Tris-HCl pH 7.3 containing protease inhibitor cocktail(Roche Diagnostic). 2 mM EDTA and 2000 units of benzonase (Merck) wereadded, cells were disrupted at 4° C. with Basic Z 0.75V Model CellDisrupter equipped with an “one shot head” (Constant System Ltd) by 2cycles, and the unbroken cells were removed by centrifugation 10 min at8 000× g at 4° C. This extract was analysed by SDS-PAGE, for comparisonwith a protein extract of the vesicles produced by ΔGKA33 bacteria. Asshown in FIG. 13, the porins PorA and PorB (identities verified byMALDI-TOF sequencing) are seen in the wild-type bacterial outer membrane(lanes 2 & 4) and also in the GNA33 knockout mutant's vesicles (lanes 3& 5). Moreover, these proteins are retained as stable trimers in thevesicles that do not dissociate into monomers in SDS-PAGE sample bufferwith a low concentration of SDS (0.2%) under seminative conditions (noheating before electrophoresis; lanes 2 & 3), but that do denature at95° C. (lanes 4 & 5).

LPS levels in detergent-extracted OMVs are typically 5-8% by weight,relative to protein [207]. When tested with the Limulus assay, theendotoxin content of the vesicles was about twice as high as found indetergent-extracted OMVs.

Finally, the yield of vesicles in a growing culture was assessed. It wasfound that up to 20 mg of OMV-associated proteins could be recovered pergram of cells (wet weight) in culture supernatants of earlyexponentially growing cultures (OD_(620 nm)=0.5).

Vesicle Immunogenicity

As the ΔmltA-derived vesicles are highly enriched in outer membraneproteins, their ability to elicit bactericidal antibodies capable ofkilling a broad panel of MenB clinical isolates was investigated.

The strain chosen for the testing was 394/98. This strain was chosenbecause it is the strain from which the MeNZB™ OMV-based vaccine isprepared, thereby aiding a direct comparison of ΔmltA vesicles of theinvention with OMVs prepared from the wild-type strain by typical priorart methods.

10 μg of each type of vesicle was adsorbed to an aluminium hydroxideadjuvant (3 mg/ml) and injected into mice 5-week old CD1 female mice(5-10 mice per group). The vesicles were given intraperitoneally on days0 and 21. Blood samples for analysis were taken on day 34, and weretested for SBA against 15 different serogroup B strains corresponding to11 different sub-types, including the four major hypervirulent lineages,using pooled baby rabbit serum as the complement source. Serumbactericidal titers were defined as the serum dilution resulting in 50%decrease in colony forming units (CFU) per ml after 60 minutesincubation of bacteria with reaction mixture, compared to control CFUper ml at time 0. Typically, bacteria incubated with the negativecontrol antibody in the presence of complement showed a 150 to 200%increase in CFU/ml during the 60 min incubation. Titers were as follows,expressed as the reciprocal of the serum dilution yielding=50% bacterialkilling:

BCA titer Serogroup:Type:Subtype mOMVs DOMVs B:4:P1.4 >8192 >32768B:15:P1.7, 4 >65536 32768 B:4, 7:P1.7, 4 >32768 >32768B:14:P1.4 >32768 >65536 B:4:P1.7, 4 >32768 8192 B:4,:P1.4 >8192 >8192B:14:P1.13 16384 512 B:4, 7:P1.7, 13 >8192 128 B:4:P1.15 >8192 128B:21:P1.9 >8192 <16 B:2b:P1.10 1024 <16 B:4, 7:P1.19, 15 1024 <16B:2b:P1.5, 2 1024 <16 B:2a:P1.2 <16 <16 B:NT:P1.3 <16 <16

The results show that serum from ΔmltA-derived vesicles were at least asbactericidally effective, and usually better than, OMVs prepared bychemical extraction, except for the homologous strain. The vesicles ofthe invention thus give much better cross-strain reactivity than typicalOMVs. Moreover, taking a 1:1024 dilution as the threshold forbactericidal efficacy, the vesicles of the invention were effectiveagainst 87% of the strains, whereas the artificial OMVs were only 40%effective.

Thus mOMVs are better than DOMVs for eliciting complement-dependentantibody killing when tested over a panel of 15 different serogroup Bstrains. The anti-mOMV mouse sera showed high bactericidal activitiesagainst the homologous strain and against 14 additional strains,including 10 different PorA subtypes. In contrast, mouse sera raisedagainst DOMVs show high bactericidal titers only against six MenBstrains, belonging to two PorA subtypes. These results indicate that theprotection of anti-mOMV sera was not only due to the elicitation ofbactericidal antibodies against PorA, which is one of the most abundantouter membrane proteins and the most potent inducer of bactericidalantibodies, but also to other bactericidal antigens which in mOMVs arepresent in higher amounts than in DOMVs.

Western Blot

To confirm that the ΔmltA-derived vesicles do contain conserved,protective antigens, they were run on an SDS-PAGE, transferred onto aPDF filter and immunoblotted using specific anti-sera against sixproteins antigens previously shown to be protective and highlyconserved, including ‘287’, ‘953’, ‘741’ (GNA1870) and ‘NadA’.

The vesicles were separated onto 10% acrylamide SDS-PAGE gels employinga Mini-Protean II electrophoresis apparatus (Bio-Rad). After proteinseparation, gels were equilibrated with 48 mM Tris-HCl, 39 mM glycine,pH 9.0, 20% (v/v) methanol and transferred to a nitrocellulose membrane(Bio-Rad) using a Trans-Blot™ semi-dry electrophoretic transfer cell.The nitrocellulose membranes were blocked with 10% (w/v) skimmed milk inPBS containing 0.2% (w/v) sodium azide.

As shown in FIG. 5, all six proteins were abundant in the vesicles. Incontrast, the same six proteins were poorly represented in the DOMVs.

In conclusion, the ΔmltA-derived vesicles are predominantly constitutedby outer membrane proteins, whereas DOMVs are heavily contaminated bycytoplasmic proteins. When used to immunize mice, sera raised againstΔmltA-derived vesicles showed a higher and wider strain coverage thanDOMVs.

Extraintestinal Pathogenic E.coli

A knockout strain of ExPEC CFT073 was prepared by isogenic deletion ofthe tolR gene, replacing it with a kanamycin resistance marker. Theknockout strain was grown to OD_(600 nm) 0.4, and the culture was thencentrifuged. The supernatant was filtered through a 0.22 μm filter andthe filtrate was precipitated using TCA. The pellet was then resuspendedin Tris buffer.

The same growth and purification procedure was used for the parentstrain, without the knockout, and SDS-PAGE analysis of the two finalpreparations is shown in FIG. 16. The right-hand band is from theknockout strain and shows enrichment of several protein bands.

Further tolR knockout ExPEC strains were prepared from strains DH5a, 536and IHE3034. Vesicles were prepared as before, and SDS-PAGE analysis ofTCA precipitates is shown in FIG. 17.

The knockout mutant produces high amounts of vesicles, and thesevesicles were subjected to proteomic analyses, including 1D and 2DSDS-PAGE and tryptic digestion of surface-exposed proteins in thevesicles followed by sequence analysis of released peptides.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

TABLE 1 NMB Protein name/theoretical MW/theoretical pl/gravy index 1 d3-10 Psort 1 NMB0018 pilin PilE/15 246/9.21/−0.571 X OM-PS 2 NMB0035conserved hypothetical protein/40 218/4.74/−0.371 X OM-IN 3 NMB0044peptide methionine sulfoxide reductase/55 718/6.54/−0.569 X OM-IN 4NMB0086 hypothetical protein/34 987/4.82/−0.505 X OM-IN 5 NMB0088 outermembrane protein P1, putative/45 902/9.35/−0.428 X OM-PS 6 NMB0109conserved hypothetical protein/43 188/6.77/−0.587 X X OM-PS(b) 7 NMB0124translation elongation factor TU/42 909/5.07/−0.136 X cyto NMB0139translation elongation factor TU/42 925/5.07/−0.142 cyto 8 NMB0138elongation factor G (EF-G)/77 244/5.08/−0.293 X cyto 9 NMB0181 outermembrane protein OmpH, putative/16 829/9.07/−0.897 X OM-PS 10 NMB0182outer membrane protein Omp85/86 254/8.37/−0.505 X X OM-PS 11 NMB0204lipoprotein, putative/12 207/8.08/−0.446 X OM-PS 12 NMB0278 thiol:disulfide interchange protein DsbA/23 428/5.16/−0.298 X OM-IN 13 NMB0281peptidyl-prolyl cis-trans isomerase/35 248/9.62/−0.388 X OM-PS 14NMB0294 thiol: disulfide interchange protein DsbA/23 566/5.09/−0.477 XOM-IN 15 NMB0313 lipoprotein, putative/52 645/9.97/−0.824 X OM-PS 16NMB0345 cell-binding factor, putative/29 448/9.13/−0.570 X X OM-PS 17NMB0346 hypothetical protein/26439/5.15/−0.716 X OM-PS 18 NMB0382 outermembrane protein class 4/23 969/6.26/−0.456 X X OM-PS 19 NMB0407 thiol:disulfide interchange protein DsbA/21 721/9.23/−0.308 X OM-PS 20 NMB0460transferrin-binding protein 2/75 292/5.79/−0.982 X OM-IN 21 NMB0461transferrin-binding protein 1/99 314/9.45/−0.699 X OM-PS 22 NMB0550thiol: disulfide interchange protein DsbC/26 451/6.93/−0.345 X OM-IN 23NMB0554 dnaK protein/68 792/4.85/−0.357 X cyto 24 NMB0622 outer membranelipoprotein carrier protein/19 996/9.47/−0.490 X OM-PS 25 NMB0623spermidine/putrescine ABC transporter/39 511/5.38/−0.437 X OM-PS 26NMB0634 iron(III) ABC transporter, periplasmic binding protein/35806/9.60/−0.338 X OM-PS 27 NMB0663 outer membrane protein NsgA/16563/9.49/−0.214 X OM-PS 28 NMB0700 IgA-specific serine endopeptidase XOM-PS 29 NMB0703 competence lipoprotein ComL/29 275/8.72/−0.761 X OM-IN30 NMB0783 conserved hypothetical protein/15 029/7.05/−0.221 X OM-PS 31NMB0787 amino acid ABC transporter/26 995/5.42/−0.287 X OM-IN 32 NMB0873outer membrane lipoprotein LolB, putative/19 575/5.23/−0.470 X OM-IN 33NMB0928 hypothetical protein/39 502/9.13/−0.596 X X OM-IN(b) 34 NMB1030conserved hypothetical protein/18 700/7.16/−0.429 X OM-PS 35 NMB1053class 5 outer membrane protein/28 009/9.68/−0.610 X X OM-PS 36 NMB1057gamma-glutamyltranspeptidase/61 590/5.94/−0.160 X OM-IN(b) 37 NMB1126hypothetical protein/22 025/8.03/−0.355 X X OM-IN NMB1164 hypotheticalprotein/22 025/8.03/−0.355 OM-IN 38 NMB1285 enolase/46 134/4.78/−0.200 Xcyto 39 NMB1301 30S ribosomal protein S1/61 177/4.9/−0.240 X cyto 40NMB1332 carboxy-terminal peptidase/53 238/9.12/−0.420 X IN 41 NMB1352hypothetical protein/13 699/9.52/−1.397 X OM-PS 42 NMB1429 outermembrane protein PorA/40.129/8.73 X X OM-PS 43 NMB1457 transketolase/71659/5.45/−0.183 X cyto 44 NMB1483 lipoprotein NlpD, putative/40947/9.55/−0.266 X X OM-PS 45 NMB1533 H.8 outer membrane protein/18886/4.61/17 X OM-IN 46 NMB1557 conserved hypothetical protein/15419/7.34/−0.429 X OM-PS 47 NMB1567 macrophage infectivity/potentiator/26875/5.50/−0.540 X OM-IN 48 NMB1578 conserved hypothetical protein/21135/4.86/−0.381 X OM-IN 49 NMB1612 amino acid ABC transporter/27970/4.87/−0.408 X OM-PS 50 NMB1636 opacity protein: authenticframeshift/27180/9.52 X X OM-PS 51 NMB1710 glutamate dehydrogenase,NADP-specific/48 490/5.98/−0.190 X cyto 52 NMB1714 multidrug efflux pumpchannel protein/48 482/8.38/−0.261 X OM 53 NMB1870 hypotheticalprotein/26 964/7.23/−0.485 X OM-IN(b) 54 NMB1898 lipoprotein/17155/7.01/−0.709 X OM-IN 55 NMB1946 outer membrane lipoprotein/29258/5.01/−0354 X OM 56 NMB1949 soluble lytic murein transglycosylase;putative/65 617/9.31/−0.525 X OM-IN 57 NMB1961 VacJ-relatedprotein/27.299/4.65/−0.344 X OM-PS 58 NMB1969 © serotype-1-specificantigen, putative X cyto 59 NMB1972 chaperonin, 60 kDa/57 423/4.9/−0.052X cyto 60 NMB1988 Iron-regulated outer membrane protein FrpB/76823/9.42/−0.700 X OM-PS 61 NMB2039 major outer membrane protein PIB/33786/6.54/−0.468 X X OM-PS 62 NMB2091 hemolysin, putative/19412/9.55/−0.152 X OM-IN 63 NMB2095 adhesin complex protein, putative/11385/9.52/−0.470 X OM-IN 64 NMB2102 elongation factor TS (EF-TS)/30330/5.30/−0.016 X cyto 65 NMB2159 glyceraldehyde 3-phosphatedehydrogenase/35 845/5.40/−0.028 X cyto

TABLE 2 NMB ANNOTATION PSORT 1 D 2 D 1 NMB0035 conserved hypotheticalprotein OM-IM X 2 NMB0044 peptide methionine sulfoxide reductase OM-IM X3 NMB0086 hypothetical protein OM-IM X 4 NMB0088 outer membrane proteinP1, putative OM-PS X X 5 NMB0109 conserved hypothetical protein OM-PS(b)X X 6 NMB0124 cyto(c, x) X X 7 NMB0138 elongation factor G (EF-G) cyto(x) X 8 NMB0182 outer membrane protein Omp85 OM-PS X X 9 NMB0204lipoprotein, putative OM-PS X 10 NMB0278 thiol: disulfide Interchangeprotein DsbA OM-IM X 11 NMB0294 thiol: disulfide Interchange proteinDsbA OM-IM X 12 NMB0313 lipoprotein, putative OM X 13 NMB0345cell-binding factor, putative OM-PS X X 14 NMB0346 hypothetical proteinOM-PS X X 15 NMB0382 outer membrane protein class 4 OM-PS X X 16 NMB0460transferrin-binding protein 2 OM-IM X 17 NMB0461 transferrin-bindingprotein 1 OM-PS X 18 NMB0462 spermidine/putrescine ABC transporter,periplasmic spermidine/putrescine-binding protein OM-PS(b) X 19 NMB0550thiol: disulfide interchange protein DsbC OM-IM X X 20 NMB0554 dnaKprotein LITT. X 21 NMB0604 alcohol dehydrogenase, zinc-containing IM X22 NMB0623 spermidine/putrescine ABC transporter, periplasmicspermidine/putrescine-binding protein OM-IM X 23 NMB0631 phosphateacetyltransferase Pta IM X 24 NMB0634 iron(III) ABC transporter,periplasmic binding protein OM-PS X 25 NMB0663 outer membrane proteinNspA OM-PS X X 26 NMB0669 conserved hypothetical protein OM-PS X 27NMB0703 competence lipoprotein ComL comL OM-IM X X 28 NMB0787 amino acidABC transporter, periplasmic amino acid-binding protein OM X 29 NMB0872conserved hypothetical protein OM-PS X 30 NMB0873 outer membranelipoprotein LolB, putative OM-IM X X 31 NMB0928 hypothetical proteinOM-IM(b) X X 32 NMB09445-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase IMX 33 NMB0983 phosphoribosylaminolNidazolecarboxamideformyltransferase/INP cyclohydrolase IM X 34 NMB1030 conservedhypothetical protein OM-PS X X 35 NMB1040 hypothetical protein OM-PS X36 NMB1053 class 5 outer membrane protein opc OM-PS X X 37 NMB1057gamma-glutamyltranspeptidase OM-IM(b) X 38 NMB1124 hypothetical proteinOM-IM X 39 NMB1125 hypothetical protein OM-IM X X 40 NMB1126hypothetical protein OM-IM X X 41 NMB1285 Enolase LITT. X 42 NMB1301 30Sribosomal protein S1 LITT. X 43 NMB1309 Nbrial biogenesis and twitchingmotility protein, putative IM X X 44 NMB1313 trigger factor FACS+ X 45NMB1332 carboxy-terminal peptidase IM X X 46 NMB1398 Cu—Zn-superoxidedismutase OM-PS X 47 NMB1429 outer membrane protein PorA porA OM-PS X X48 NMB1483 lipoprotein NlpD OM-PS X X 49 NMB1497 TonB-dependent receptorOM X 50 NMB1518 acetate kinase IM X 51 NMB1533 H.8 outer membraneprotein OM-PS X 52 NMB1567 macrophage infectivity potentiator OM-IM X 53NMB1574 ketol-acid reductoisomerase CYTO X 54 NMB1612 amino acid ABCtransporter, periplasmic amino acid-binding protein OM-IM X 55 NMB1710glutamate dehydrogenase, NADP-specific LITT. X 56 NMB1812 putative, pilQprotein, authentic frameshift OM-PS X 57 NMB1870 hypothetical proteinOM-IM(b) X 58 NMB1898 lipoprotein mlp OM-IM X X 59 NM81902 DNApolymerase III, beta subunit CYTO X 60 NMB1949 soluble lytic mureintransglycosylase, putative OM-IM X 61 NMB1961 VacJ-related-protein OM-PSX 62 NMB1972 chaperonin, 60 kDa LITT. X X 63 NMB1988 iron-regulatedouter membrane protein FrpB OM-PS X X 64 NMB2039 major outer membraneprotein PIB OM-PS X X 65 NMB2091 hemolysin, putative OM-IM X 66 NMB2139conserved hypothetical protein OM-IM X 34 56

TABLE 3 NMB0035 NMB0044 NMB0086 NMB0088 NMB0109 NMB0124 NMB0138 NMB0182NMB0204 NMB0278 NMB0294 NMB0313 NMB0345 NMB0346 NMB0382 NMB0460 NMB0461NMB0550 NMB0554 NMB0623 NMB0634 NMB0663 NMB0703 NMB0787 NMB0873 NMB0928NMB1030 NMB1053 NMB1057 NMB1126 NMB1285 NMB1301 NMB1332 NMB1429 NMB1483NMB1533 NMB1567 NMB1612 NMB1710 NMB1870 NMB1898 NMB1949 NMB1961 NMB1972NMB1988 NMB2039 NMB2091

TABLE 4 NMB0044 NMB0086 NMB0204 NMB0278 NMB0294 NMB0313 NMB0345 NMB0346NMB0460 NMB0550 NMB0873 NMB0928 NMB1030 NMB1057 NMB1483 NMB1870 NMB1898NMB1961 NMB2091

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What is claimed is:
 1. A process for preparing Escherichia bacterialvesicles, comprising the steps of: (i) culturing an Escherichiabacterium in a culture medium such that the bacterium releases vesiclesinto said medium; and (ii) collecting the vesicles from said medium,wherein: (a) the bacterium has a cell wall that includes peptidoglycan;and (b) the bacterium has a knockout mutation of its mltA gene.
 2. Theprocess of claim 1, wherein the bacterium also has a knockout mutationof at least one further gene.
 3. The process of claim 1, wherein theEscherichia bacterium is a pathogenic Escherichia coli (E.coli)bacterium.
 4. The process of claim 3, wherein the pathogenic E.coli isan extraintestinal pathogenic bacterium, a uropathogenic bacterium, or ameningitis/sepsis-associated bacterium.
 5. The process of claim 2,wherein the bacterium is E.coli.
 6. The process of claim 5, wherein thebacterium is a pathogenic E.coli.
 7. The process of claim 6, wherein thepathogenic E.coli is an extraintestinal pathogenic bacterium, auropathogenic bacterium, or a meningitis/sepsis-associated bacterium. 8.The process of claim 1, wherein the bacterium is a pathogenicEscherichia coli bacterium, which does not express a protein of theTol-Pal complex.
 9. The process of claim 8, wherein the E.coli bacteriumis a tolR-strain.